In laser microdissection, selected tissue regions or cells are separated out or cut out (“dissected”) from microscopic tissue specimens with the aid of a focused laser beam for further analysis.
DE 100 18 253 C2, DE 10 2005 028 062 B4 and DE 10 2005 008 925 A1 by the applicant describe possible methods and systems for laser microdissection employing a microscope.
Capillary-based analytical apparatuses are so far advanced that individual cells or even chromosomes detached from tissue can be transported in droplets or solution through capillary systems, and sorted, analysed and also utilised further after analyses. Capillary-based analytical techniques are used and promoted, for example, by FLUIDIGM and RAINDANCE TECHNOLOGIES.
In order to be able to be analysed using these techniques, the specimens and individual cells must first be separated; this can be effected by fluorescence-based flow cytometry, laser microdissection and transpipetting into an appropriate feed medium for the capillary systems.
Various collectors are available for so-called “dissectates” cut out in the laser microdissection, for example so-called “tube caps”, 8-well strips, 8-well strip caps or so-called “lab coat pocket laboratories” (also called “lab on a chip”), for example AmpliGrid devices. After the laser microdissection process, these collectors are removed physically from the dissection machine and further processed separately. As a rule centrifugation and pipetting steps are necessary for this, before the dissectate can be fed in appropriate solution to an analytical method, such as the polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR) or real time or quantitative PCR (rt-/qPCR) or mass spectrometry (MS).
This procedure has the disadvantage that the possibilities for an automated further processing of specimens directly after isolation by means of laser microdissection are severely limited. Furthermore, the necessary complex centrifugation and pipetting steps have a high risk of contamination and specimen loss.
EP 2 083 257 A1 discloses a method and a device for transferring a microscopic, isolated specimen. Here, a nanosuction apparatus is positioned over a specimen which has been cut out of a specimen body on an object table. The nanosuction apparatus actively sucks up the specimen from the object table, before it is swivelled and then blows out the specimen again at a different place. The specimen can thus be transferred, for example, into a reaction vessel. Before the sucking up, however, visual inspection of the specimen is not possible or is at least only inadequate, because this is still surrounded by the remaining specimen body from which it has been cut out.
DE 10 2009 016 512 B4 discloses a laser ablation chamber consisting of a container which can be closed in a gas-tight manner from the top with a cover glass, on the underneath of which the specimen to be analysed is located. The container has a feed line and a removal line for a transporting gas (for example argon). An examination of the specimen is first carried out with a determination of the regions to be removed by means of laser ablation. This examination is carried out with a laser microdissection apparatus utilising the high-resolution lens of such an apparatus. After determination of appropriate regions, these are detached from the specimen by means of laser ablation and transported out of the gas-tight container, for example into an analytical device, such as a mass spectrometer, by feeding in the transporting gas. Further specimen regions can be analysed, depending on the result of the analysis. The known laser microdissection can be employed for this, which allows dissection of specimen sections in the submicron range with high precision. For laser microdissection, the laser ablation chamber described is replaced by conventional specimen holders for laser microdissection.
The object of DE 103 29 674 A1 is to overcome the disadvantages of UV ablation, such as the absorption of UV radiation in biological tissue or the low cutting quality. For cutting biological material, the use of ultra-short laser pulses in the pico- or femtosecond range with secondary frequencies of at least 1,000 Hz at a laser wavelength of greater than 400 nm (near infra-red) is proposed here, the laser being focused on the specimen in order to generate high intensities of at least 1012 W/cm2 there. By this means a so-called non-resonant multiphoton absorption is achieved, which generates a plasma for the cutting action. In one embodiment the biological material cut out is separated from the surrounding specimen by the gravitational effect and falls onto a cover glass coated with lysine. In this procedure, for example, the specimen with the film carrying it is applied to the underside of a glass window. A silicone ring joins the glass window to the lysine-coated cover glass. The biological material cut out can be either sucked off, for example by inserting two cannulas through the silicone ring, or flushed out by means of hydrodynamic flow and collected by means of a sterile filter. The upper glass window, the silicone ring and the lower cover glass for collecting the material cut out overall form a closed sterile chamber.